It is well established that angiogenesis plays an important role in tumor progression and metastasis and anti-angiogenesis represents a clinically validated anti-cancer strategy (Folkman, J., Nat Med 1, 27-31 (1995); Ferrara, N. and Kerbel, R. S., Nature 438, 967-974 (2005); Carmeliet, P. Nat Med 9, 653-660 (2003)). Angiogenesis also plays a key pathogenic role in a variety of other disorders, including age-related macular degeneration (AMD). Choroidal neovascularization has been reported to be dependent, at least in part, on neutrophil infiltration (Zhou et al., Mol Vis 11:414-424 (2005)). Tumor cells have been traditionally considered the main source of mediators of angiogenesis (Folkman, J., N Engl J Med 385, 1182-1186 (1971)). Indeed, much research has shown that cancer cells may produce a variety of angiogenic factors, including vascular endothelial growth factor-A (VEGF-A), angiopoietins, hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), and various mutations in oncogenes or tumor suppressor genes may result in increased production of at least some of these factors (Rak, J., et al., Cancer Res 55, 4575-4580 (1995); Wizigmann, et al., Cancer Res 55, 1358-1364 (1995)). However, compelling evidence now supports the notion that the stroma, consisting of fibroblasts, pericytes, mesenchymal stem cells and inflammatory-immune cells, and endothelial progenitors also contribute to tumor growth not only through secretion of angiogenic factors but also by modulation of the immune system (Hanahan, D. and Weinberg, R. A., Cell 100, 57-70 (2000); Coussens, L. M. and Werb, Z., Nature 420, 860-867 (2002); Blankenstein T., Curr Opin Immunol 17:180-186 (2005); Karnoub et al., Nature 449:557-563 (2005); Orimo et al., Cell Cycle 5:1497-1601 (2006); and Rafii, S. et al., Nat Rev Cancer 2, 826-835 (2002)). Potentially, some of these cells may inhibit tumor growth by immune surveillance mechanisms, but much of the evidence indicates that a marked infiltration by leukocytes and other inflammatory cells in tumors carries a poor prognosis (Coussens, et al., supra).
Recent studies have directly implicated different populations of myeloid cells in the regulation of tumor angiogenesis (Da Palma, M., et al., Nat Med 9, 789-795 (2003); Yang, L. et al., Cancer Cell 6, 409-421 (2004); De Palma M., et al., Cancer Cell 8, 211-226 (2005)) and VEGF-induced neovascularization in the adult (Grunewald, M. et al., Cell 124, 175-189 (2006)). Very recent studies have provided evidence for a role of CD11b+Gr1+ myeloid cells in mediating refractoriness to anti-VEGF therapy in some tumor models (Shojaei, F. et al., Cell 124, 175-189 (2006)). The role of neutrophils in initiating the angiogenic switch in a transgenic model of multi-stage carcinogenesis has been described (Nozawa, H. et al., Proc Natl Acad Sci USA 103, 12493-12498 (2006)). Myeloid cells may locally secrete angiogenic factors or produce proteases such as matrix metalloproteinase-9 (Yang, L., et al., supra; Nozawa et al., supra; van Kempen, L. C. et al., Eur J Cancer 42, 728-734 (2006)), which may in turn promote angiogenesis by increasing the bioavailability and activity of VEGF-A in the tumor microenvironment (Bergers G., et al., Nat Cell Biol 2, 737-744 (2000)). Nevertheless, our understandings of the mechanisms by which myeloid cells are mobilized from the BM and promote tumorigenesis remains incomplete.
Bv8 and EG-VEGF are two highly related secreted proteins, also referred to as prokineticin-1 and -2, which structurally belong to a larger class of peptides defined by a five disulphide bridge motif called a colipase fold (DeCouter, J. et al., Nature 420, 860-867 (2002); LeCouter. J. et al., Proc Natl Acad Sci USA 100, 2685-2690 (2003); Li, M. et al., Mol Pharmacol 59, 692-698 (2001)). Bv8 was initially identified as a secreted protein from the skin of the frog Bombina variegate (Mollay, C. et al., Eur J Pharmacol 374, 189-196 (1999)). The cloning and expression of Bv8 are described in WO 03/020892 published on Mar. 13, 2003. Bv8 and EG-VEGF bind two highly related G-protein coupled receptors (GPCR), EG-VEGF/PKR-1 (R1) and EG-VEGF/PKR-2 (R2) (Masuda, Y et al., Biochem Biophys Res Commun 293, 496-402 (2002); Lin, D. C. et al., J Biol Chem 277, 19276-19280 (2002)). EG-VEGF and Bv8 were characterized as mitogens selective for specific endothelial cell types (LeCouter, J. et al., (2001) and (2003), supra). Other activities have been ascribed to this family, including nociception (Mollay, C. et al., supra), gastrointestinal tract motility (Li, M. et al., supra), regulation of circadian locomotor rhythm (Cheng, M. Y., et al., Nature 417, 405-410 (2002)) and olfactory bulb neurogenesis (Matsumoto, S., et al., Proc Natl Acad Sci USA 103, 4140-4145 (2006)). Furthermore, Bv8 or EG-VEGF stimulated production of granulocytic and monocytic colonies in vitro (LeCouter, J. et al., (2003), supra; Dorsch, M. et al., J. Leukoc Biol 78(2), 426-34 (2005)). Bv8 has been characterized as a chemoattractact for macrophages (LeCouter et al., Proc Natl Acad Sci USA 101, 16813-16919 (2004)).
Recognition of vascular endothelial growth factor (VEGF) as a primary regulator of angiogenesis in pathological conditions has led to numerous attempts to block VEGF activities. VEGF is one of the best characterized and most potent positive regulators of angiogenesis. See, e.g., Ferrara, N. & Kerbel, R. S. Angiogenesis as a therapeutic target. Nature 438:967-74 (2005). In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF, as a pleiotropic growth factor, exhibits multiple biological effects in other physiological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx. Ferrara and Davis-Smyth (1997) Endocrine Rev. 18:4-25. Moreover, studies have reported mitogenic effects of VEGF on a few non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic duct cells and Schwann cells. See, e.g., Guerrin et al. J. Cell Physiol. 164:385-394 (1995); Oberg-Welsh et al. Mol. Cell. Endocrinol. 126:125-132 (1997); and, Sondell et al. J. Neurosci. 19:5731-5740 (1999).
Recognition of vascular endothelial growth factor (VEGF) as a primary regulator of angiogenesis in pathological conditions has led to numerous attempts to block VEGF activities. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been proposed for use in interfering with VEGF signaling. See, e.g., Siemeister et al. Cancer Metastasis Rev. 17:241-248 (1998). Anti-VEGF neutralizing antibodies have been shown to suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al. Nature 362:841-844 (1993); Warren et al. J. Clin. Invest. 95:1789-1797 (1995); Borgström et al. Cancer Res. 56:4032-4039 (1996); and Melnyk et al. Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders (Adamis et al. Arch. Opthalmol. 114:66-71 (1996)). Indeed, a humanized anti-VEGF antibody, bevacizumab (AVASTIN®, Genentech, South San Francisco, Calif.) has been approved by the US FDA in combination with intravenous 5-fluorouracil-based (5-FU) chemotherapy, for first- or second-line treatment of patients with metastatic carcinoma of the colon or rectum and in combination with carboplatin and paclitaxel for the first-line treatment of patients with unresectable, locally advanced, recurrent or metastatic non-squamous non-small cell lung cancer (NSCLC). See, e.g., Ferrara et al., Nature Reviews Drug Discovery, 3:391-400 (2004).
However, the long-term ability of therapeutic compounds to interfere with tumor growth is frequently limited by the development of drug resistance. Several mechanisms of resistance to various cytotoxic compounds have been identified and functionally characterized, primarily in unicellular tumor models. See, e.g., Longley, D. B. & Johnston, P. G. Molecular mechanisms of drug resistance. J Pathol 205:275-92 (2005). In addition, host stromal-tumor cell interactions may be involved in drug-resistant phenotypes. Stromal cells secrete a variety of pro-angiogenic factors and are not prone to the same genetic instability and increases in mutation rate as tumor cells (Kerbel, R. S. Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. Bioessays 13:31-6 (1991). Reviewed by Ferrara & Kerbel and Hazlehurst et al. in Ferrara, N. & Kerbel, R. S. Angiogenesis as a therapeutic target. Nature 438:967-74 (2005); and, Hazlehurst, L. A., Landowski, T. H. & Dalton, W. S. Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death. Oncogene 22:7396-402 (2003).
In preclinical models, VEGF signaling blockade with the humanized monoclonal antibody bevacizumab (AVASTIN®, Genentech, South San Francisco, Calif.) or the murine precursor to bevacizumab (A4.6.1 (hybridoma cell line producing A4.6.1 deposited on Mar. 29, 1991, ATCC HB-10709)) significantly inhibited tumor growth and reduced tumor angiogenesis in most xenograft models tested (reviewed by Gerber & Ferrara in Gerber, H. P. & Ferrara, N. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cancer Res 65:671-80 (2005)). The pharmacologic effects of single-agent anti-VEGF treatment were most pronounced when treatment was started in the early stages of tumor growth. If treatment was delayed until tumors were well established, the inhibitory effects were typically transient, and tumors eventually developed resistance. See, e.g., Klement, G. et al. Differences in therapeutic indexes of combination metronomic chemotherapy and an anti-VEGFR-2 antibody in multidrug-resistant human breast cancer xenografts. Clin Cancer Res 8:221-32 (2002). The cellular and molecular events underlying such resistance to anti-VEGF treatment are complex. See, e.g., Casanovas, O., Hicklin, D. J., Bergers, G. & Hanahan, D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8:299-309 (2005); and, Kerbel, R. S. et al. Possible mechanisms of acquired resistance to anti-angiogenic drugs: implications for the use of combination therapy approaches. Cancer Metastasis Rev 20:79-86 (2001). A variety of factors may be involved. For example, combination treatment with compounds targeting VEGF and fibroblast growth factor (FGF) signaling improved efficacy and delayed onset of resistance in late-stage tumors in a genetic model of pancreatic islet carcinogenesis. See, Casanovas, O., Hicklin, D. J., Bergers, G. & Hanahan, D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8, 299-309 (2005). Other investigators have identified tumor-infiltrating stromal fibroblasts as a potent source of alternative pro-angiogenic factors. See, e.g., Dong, J. et al. VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis. Embo J 23:2800-10 (2004); and, Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121:335-48 (2005).
Inflammatory cells can participate in angiogenesis by secreting inflammatory cytokines, which can affect endothelial cell activation, proliferation, migration, and survival (reviewed in Albini et al. and Balkwill et al. in Albini, A., Tosetti, F., Benelli, R. & Noonan, D. M. Tumor inflammatory angiogenesis and its chemoprevention. Cancer Res 65:10637-41 (2005); and, Balkwill, F., Charles, K. A. & Mantovani, A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7:211-7 (2005). Several tumor-infiltrating inflammatory cells secrete pro-angiogenic factors, including monocytes/macrophages (see, e.g., De Palma, M. et al. Tie2 identities a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8:211-26 (2005); and, Yang, L. et al. Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6:409-21 (2004)), T- and B-lymphocytes (see, e.g., Freeman, M. R. et al. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 55:4140-5 (1995)), vascular leukocytes (see, e.g., Conejo-Garcia, J. R. et al. Vascular leukocytes contribute to tumor vascularization. Blood 105:679-81 (2005)), dendritic cells (see, e.g., Conejo-Garcia, J. R. et al. Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Nat Med 10:950-8 (2004)), neutrophils (see, e.g., Coussens, L. M., Tinkle, C. L., Hanahan, D. & Werb, Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103:481-90 (2000)), and mast cells (see, e.g., Coussens, L. M. et al. Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev 13:382-97 (1999); and (reviewed in de Visser and Coussens in de Visser, K. E., Eichten, A. & Coussens, L. M. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 6:24-37 (2006)). It was suggested that bone marrow-derived endothelial progenitor cells (EPCs (see, e.g., Lyden, D. et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 7, 1194-201 (2001)) and perivascular progenitor cells (see, e.g., Song, S., Ewald, A. J., Stallcup, W., Werb, Z. & Bergers, G. PDGFRbeta+ progenitor cells in tumors regulate pericyte differentiation and vascular survival. Nat Cell Biol 7:870-9 (2005)) contribute to vessel formation in some experimental models of tumor growth (reviewed in Rafii et al. in Rafii, S. Lyden, D., Benezra, R., Hattori, K. & Heissig, B. Vascular and haematopoietic stem cells: novel tar sets for anti-angiogenesis therapy? Nat Rev Cancer 2:826-35 (2002)). Myeloid lineage hematopoietic cells, including tumor-associated macrophages (TAMS), were shown to stimulate angiogenesis either directly by secreting angiogenic factors or indirectly by producing extracellular matrix-degrading proteases, which in turn release sequestered angiogenic factors (reviewed in Lewis, C. E. & Pollard, J. W. Distinct role of macrophages in different tumor microenvironments. Cancer Research 66:605-612 (2006); and, Naldini, A. & Carraro, F. Role of inflammatory mediators in angiogenesis. Curr Drug Targets Inflamm Allergy 4:3-8 (2005)). Among the myeloid cell lineages, CD11b+Gr1+ progenitor cells isolated from the spleens of tumor-bearing mice promoted angiogenesis when co-injected with tumor cells (see, e.g., Yang, L. et al. Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6:409-21 (2004)) and tumor-infiltrating macrophage numbers correlated with poor prognosis in some human tumors (reviewed in Balkwill et al. in Balkwill, F., Charles, K. A. & Mantovani, A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7:211-7 (2005)). However, in another study, macrophages inhibited growth of experimental tumors in mice, suggesting their potential as anticancer therapy. See, e.g., Kohchi, C. et al. Utilization of macrophages in anticancer therapy: the macrophage network theory. Anticancer Res 24:3311-20 (2004). Shojaei, F. Wu, et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD11b(+)Gr1(+) myeloid cells. Nat Biotechnology 2007 25(8):911-20, reported on the role of CD11b(+)Gr1(+) myeloid cells in the resistance of tumors to treatment with anti-VEGF antibodies. Similar findings are disclosed in co-pending U.S. application Ser. No. 11/692,681, filed on Mar. 28, 2007.
Despite the relative abundance of myeloid cells and their potential to produce pro-angiogenic factors, their role in tumor resistance to anti-VEGF treatment remains unknown. There is a need to discover and understand the biological functions of myeloid cells, resistant tumors, and the factors that they produce. The present invention addresses these and other needs, as will be apparent upon review of the following disclosure.